Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.     

Metabolism of three cyclic nitrosamines in Sprague-Dawley rats

The metabolism of three cyclic nitrosamines has been studied in Sprague-Dawley rats. The compounds were nitrosopyrrolidine, nitrosohexamethyleneimine, and nitrosohepatamethyleneimine and were labeled at the alpha carbon with 14C. At low doses (2 to 4 mg/animal) the compounds were metabolized to 14CO2 to the extent of 77, 43, and 27%, respectively, after 24 hr. At doses closer to the 50% lethal dose of the compounds (70 to 160 mg/animal) the metabolism values were only 14, 4, and 8%, respectively, after 24 hr. The significance of these results is discussed.     

Anti-inflammatory agents and allergen-induced beta2-receptor dysfunction in isolated human bronchi

Antigen challenge causes beta2-adrenoceptor dysfunction in sensitized human bronchi (Am. J. Respir. Crit. Care Med. 1997;155:1230-1234). This study investigated whether the dysfunction can be prevented by anti-inflammatory agents. Human bronchial rings (2 to 4 mm) from surgery were passively sensitized to house dust mite and challenged (1) with allergen only, (2) with allergen plus indomethacin (10(-)5 M), (3) with allergen plus nedocromil sodium (10(-)7 M to 10(-)5 M), (4) with allergen plus the H1-receptor antagonist cetirizine (10(-)7 M to 10(-)5 M), and (5) with allergen plus the peptido-leukotriene receptor antagonist iralukast (10(-)7 M to 10(-)5 M). Rings were first contracted with 10(-)6 M carbachol and then relaxed with salbutamol (10(-)9 M to 10(-)4 M). The concentration-relaxation curve to salbutamol was shifted significantly to the right in the rings challenged with allergen only compared with control rings. In the rings challenged with allergen plus nedocromil sodium (10(-)6 M and 10(-)5 M) or iralukast (10(-)6 M and 10(-)5 M) the concentration-relaxation curves to salbutamol were significantly shifted to the left compared with rings challenged in saline alone, suggesting a protective effect against beta2-adrenoceptor dysfunction. Neither allergen plus cetirizine nor allergen plus indomethacin shifted significantly the concentration-relaxation curves to salbutamol compared with rings challenged in saline alone. We conclude that the release of peptido-leukotrienes may play a significant role in causing the allergen-induced beta2-receptor dysfunction in passively sensitized human bronchi.     

IgE-induced passive sensitization of human isolated bronchi and lung mast cells

Passive sensitization of human isolated lung with serum from atopic asthmatic patients provides an opportunity to study the link between airway hyper-responsiveness and the allergic process. To directly demonstrate the role of immunoglobulin E (IgE) in the effect of the atopic serum, we have compared the effect of passively sensitizing both human bronchi and isolated lung mast cells with either serum from atopic asthmatic patients or human monoclonal IgE. Peripheral bronchi ( < 5 mm in internal diameter) were dissected out from human lung obtained at thoractomy and isometric contraction was studied in response to a variety of immunological stimuli according to the sensitization protocol. Mast cells were also isolated from human lung and histamine release was measured under similar experimental conditions. A contractile response was elicited by either the specific antigen or anti-IgE (0.6-600 ng.mL-1) but not anti-immunoglobulin G (IgG) 0.2-20 micrograms.mL-1) in airways sensitized with atopic serum (total IgE concentration of approximately 1,000 international units (IU).mL-1). The maximal contractile response to anti-IgE was 75 +/- 22% of the response to 1 mM acetylcholine. Similarly, anti-IgE released histamine from isolated lung mast cells sensitized with atopic serum up to 22.4 +/- 2% of total histamine measured within mast cells. When isolated airways or mast cells were sensitized with human monoclonal IgE (1,000 IU.mL-1), response to anti-IgE in terms of contractile response or histamine release, respectively, were not significantly different from those obtained following passive sensitization with atopic serum. Finally, the bronchial contractile response to anti-IgE depended not only on the concentration of anti-IgE but also on that of IgE (300-2,000 IU.mL-1) used to sensitize the airways. These results indicate that the effect of antigen or anti-IgE in peripheral bronchi passively sensitized with atopic serum is mimicked when sensitization is carried out directly with human monoclonal IgE.     

Influence of external pH on ciliary beat frequency in human bronchi and bronchioles

To assess the efficiency of transvaginal ultrasonography (TVUS) in the screening of pelvic pathologies in the initial workup of infertile women, we carried out a prospective comparison of sonographic diagnosis with laparoscopic and pathological findings. Between February 1994 and April 1995, 133 premenopausal non-pregnant women underwent TVUS on the day before laparoscopy. The efficiency of TVUS in detecting pelvic pathologies was 90.2% with a sensitivity of 86.2%, a specificity of 97.8% and positive and negative predictive values of 98.6 and 78.8% respectively. If the six false-negative cases with a histological diagnosis of minimal endometriosis were defined as 'normal pelvis', sensitivity and specificity could be corrected to 92.5 and 98.6% respectively. Endometriomas were diagnosed by TVUS with an efficiency of 96.4%, with a sensitivity and a specificity of 90 and 96.7 % and with positive and negative predictive values of 75 and 99.1% respectively. The sensitivity of vaginal sonographic characterization of pelvic adhesions was 61.1% with a specificity and positive predictive value of 98.2 and 84.6%. The negative predictive value of TVUS was 94.1%. These data suggest that it is not possible to characterize pelvic adhesions, especially filmy adhesions, with acceptable accuracy. However, in the initial workup of infertile women, if the patient is young, if both hysterosalpingography and TVUS are negative, laparoscopy could be postponed. In couples with severe male factor infertility and for whom in-vitro fertilization or intracytoplasmic sperm injection is the treatment of choice, laparoscopy might be avoided where the TVUS is negative.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

Metabolism of 8,11,14-eicosatrienoic acid in human platelets

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).     

Metabolism of 6-mercaptopurine in human leukemic cells

The PRPP concentrations, PRPP formation, and phosphorylation of 6-mercaptopurine in leukocyte suspensions and homogenates prepared from leukemic patients were studied...     

Metabolism and bioactivation of clozapine by human liver in vitro

The metabolism of clozapine by human liver has been investigated in vitro. Irreversible protein-binding and conjunction with model nucleophiles have been used as markers for bioactivation of clozapine, while stable metabolite formation has been assessed using radiometric HPLC. In all nine liver microsomal preparations investigated, clozapine was extensively metabolized to the stable products desmethylclozapine (range 19%-27.2%), N-oxide (1.5-20.5%) and three polar metabolites (0-20.8%), and was bioactivated to a protein-reactive metabolite (0.6-2.1%). The CYP2D6 genotype did not influence the capacity of the livers to form these metabolites. All metabolic pathways were inhibited by ketoconazole, indicating the involvement of the cytochrome P450 enzymes. Isozyme-selective inhibitor studies demonstrated that whereas demethylation was performed by CYP1A2, N-oxidation and chemically reactive metabolite formation were dependent upon multiple forms of P450. The N-oxide was readily reduced back to clozapine in the presence of NADPH, this conversion being inhibited by ascorbic acid. Glutathione (1 mM) decreased covalent binding by 70%. The amount of putative adduct formed in the presence of glutathione (13.4 +/- 0.9%) was much greater than the covalent binding (mean 1.1 +/- 0.2%). The bioactivation of clozapine was, like the N-oxidation of clozapine, a reversible process. In summary, our results indicate clozapine undergoes extensive metabolism by human liver to both stable and chemically reactive metabolites, the formation of which is catalyzed by the cytochrome P450 enzymes. The role of the reactive metabolite, which may be a free radical, in the pathogenesis of clozapine agranulocytosis and hepatotoxicity requires further study.     

Allergen challenge of passively sensitized human bronchi alters M2 and beta2 receptor function

We investigated whether antigen challenge may alter M2 and beta2 receptor function in isolated passively sensitized human bronchi. Bronchial rings (2-4 mm internal diameter) were obtained from 12 patients. Passive sensitization was induced by serum containing high IgE levels to Dermatophagoides pteronyssinus and Dermatophagoides farinae. Rings from six patients were used to study M2 receptor function by incubating with cumulatively increasing concentrations (10(-8) M to 10(-4) M) of pilocarpine and applying electric field stimulation (EFS) at 24 Hz. The rings from the other six patients were used to study beta2 receptor function by precontracting with carbachol 10(-6) M and adding cumulatively salbutamol (10(-9) M to 10(-4) M). Additional rings from these patients were used to determine whether dysfunction occurred distal to cAMP production by precontracting with carbachol 10(-6) M and adding cumulatively theophylline (10(-9) M to 10(-4) M). The attenuation of EFS-induced force by pilocarpine and the relaxation of carbachol-precontracted rings by salbutamol were less in challenged than in control and sensitized rings. No difference between challenged, sensitized, and control rings was observed with theophylline. We conclude that allergen challenge in passively sensitized isolated human bronchial rings may result in M2 and beta2 receptor dysfunction without involving mechanisms distal to cAMP formation. It appears that products from inflammatory cells recruited from blood are not necessary for this receptor dysfunction.     

Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages

The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.     

Beta-lactoglobulin suppresses melanogenesis in cultured human melanocytes

Eight cases of cervical necrotizing fasciitis are presented. Three were odontogenic, two were pharyngeal in origin and three were primary or idiopathic. Soft tissue gas was recognized in four patients. The bacteriology showed streptococci on the top of the list (50%), while for the idiopathic cases, it was monomicrobial and caused by staphylococci. Third generation cephalosporin and metronidazole represent good initial empirical antibacterial coverage. Histopathologically, all cases showed extensive necrosis of the debrided fascia and vascular thrombosis of the dermal vessels. The mortality rate was 3/8 (37.5%). Early diagnosis of cervical necrotizing fasciitis and initiation of definitive therapy in an intensive care environment is essential to minimize mortality. It is also important to recognize that this devastating infection may occur spontaneously, and it should be suspected in patients with unexplained soft tissue pain and tenderness.     

Lipid synthesis in cultured human embryonic fibroblasts

We describe here the pathways by which human embryonic fibroblasts synthesize lipids. In these studies, we quantitated the phospholipds by their phosphorus content and by their acyl components. These determinations defined both the chemical composition of the cellular membranes as well as their metabolic turnover. Using radiolabeled precursors, we have shown (a) synthesis of the glycerol moiety via glycolysis and the action of glycerokinase, (b) utilization of both exogenously added and endogenously synthesized fatty acids, (c) synthesis de novo of phosphatidyl choline and phsphatidyl ethanolamine from their base precursors, and (d) the methylation of phosphatidyl ethanolamine yielding phosphatidyl choline. Dividing cells synthesized phosphoglyceride more rapidly than cells in the stationary phase. However, considerable turnover of cellular lipid did occur in the stationary phase.     

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7 +/- 8.7 nmoles per hour per milligram of protein and an apparent Km for citrulline of 2 X 10(-4) M, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7 +/- 3.8 nmoles/hr/mg and an apparent Km for citrulline of 2 X 10(-2) M. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.     

Metabolism of hydroperoxy-phospholipids in human hepatoma HepG2 cells

Two enzymatic mechanisms have been proposed for the metabolism of hydroperoxy-phospholipids: i) the combined action of phospholipase A2 and glutathione peroxidase, and/or ii) direct enzymatic reduction. The latter reaction may be catalyzed by selenium-dependent phospholipid hydroperoxide glutathione peroxidase and/or by glutathione S-transferase alpha. To study the pathway of this reaction, we used human hepatoma HepG2 cells into which was incorporated labeled, hydroperoxy-phospholipids. The major product of incorporated l-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine was the corresponding hydroxy-phospholipid with no hydroxy- or hydroperoxy-fatty acids. The contributions to reduction of hydroperoxy-phospholipids in HepG2 cells from glutathione S-transferase Al and phospholipid hydroperoxide glutathione peroxidase were calculated to be 0.5% and 99.5%, respectively. Increasing selenium in the cell culture medium led to increases in selenium-dependent phospholipid hydroperoxide glutathione peroxidase activity but not in glutathione S-transferase alpha. This increase in the selenium-dependent enzyme was paralleled by a concomitant increase in the extent of reduction of the incorporated hydroperoxy-phospholipid. We conclude that the main metabolic fate of hydroperoxy-phospholipids in HepG2 cells is by direct reduction to hydroxy-phospholipids by phospholipid hydroperoxide glutathione peroxidase but also by glutathione S-transferase alpha, and that phospholipase A2/selenium-dependent glutathione peroxidase does not play a significant role in the reduction.     

Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions

The rat liver microsome-mediated mutagenicities of a series of N-nitrosodialkylamines and heterocyclic N-nitrosamines were determined in a liquid incubation system using Salmonella typhimurium TA1530. The influence on mutation frequency of the concentration of co-factors for mixed-function oxidase and composition and molarity of the buffer was investigated, using N-nitrosomorpholine as substrate. The mutagenicity of the N-nitroso compounds in the liquid incubation system under optimal reaction conditions at equimolar concentration was compared quantitatively with that obtained in a soft-agar incorporation assay. N-Nitrosodi-n-pentylamine and N-nitrosodi-n-butylamine showed no enzyme-mediated mutagenicity in the liquid incubation system, and metabolically activated N-nitroso-dimethylamine and N-nitroso-diethylamine showed negligible mutagenic activity in the soft-agar assays. In contrast with these results with the N-nitrosodialkylamines, the mutagenic effects of heterocyclic N-nitrosamines were similar in the liquid incubation system and in soft-agar incorporation assays. The heterocyclic N-nitrosamines showed rat-liver microsome-mediated mutagenicity in the following descending order: N-nitrosomorpholine greater than N-nitrosopyrrolidine greater than N-nitrosopiperidine greater than N-nitroso-N'-methylpiperazine. Seven human liver specimens converted all heterocyclic N-nitrosamines into mutagens; this activity was similar to that of rat liver, except that for N-nitroso-N'-methylpiperazine, fractions from three human liver biopsies were three to 30 times more active than those from untreated rats. The specific reversion of S. typhimurium TA1530 to histidine prototrophy provides experimental evidence that all the N-nitrosamines studied were converted by liver microsomal enzymes into monofunctional alkylating agents.     

Metabolism of catechol estrogen by human erythrocytes

In a study designed to evaluate the kinetics of catechol estrogen formation from plasma estrone in vivo, we obtained evidence that the red blood cell (RBC) enzyme, catechol-O-methyl transferase, catalyzes the transformation of 2-hydroxyestrone to 2-methoxyestrone. Under in vitro conditions, the rate of conversion of 2-hydroxyestrone to 2-methoxyestrone by human RBC's was such that 7 nmol of 2-methoxyestrone were formed per h per ml of RBC.     

Comparison of the effects of cyclic AMP analogues on cholesterol metabolism in cultured rat and hamster hepatocytes

The effects of two cell-permeable cyclic AMP analogues, 8-chloro cyclic AMP (8-Cl cAMP) and 8-(4-chlorophenylthio) cyclic AMP (8-CPT cAMP), on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis were compared in cultured rat and hamster hepatocytes. Cholesterol esterification, as measured by the incorporation of [3H]oleate into cholesteryl ester, was increased by 58-88% by the analogues in rat hepatocytes and by 33-43% in hamster cells. The response in rat hepatocytes, however, was observed after a relatively short incubation time (28% increase after 1 hr), whereas that in hamster cells required a longer period (36% after 12 hr) to become apparent. The activity of the cytosolic neutral cholesteryl ester hydrolase in rat hepatocytes was also stimulated by both cyclic AMP analogues (31-37%, but the microsomal activity was unaffected. In hamster hepatocytes, however, microsomal cholesteryl ester hydrolase activity was increased (47-80%) in the presence of 8-Cl cAMP or 8-CPT cAMP. Bile acid synthesis was increased by 8-CPT cyclic AMP in rat cells (approximately 25%) but was unchanged by both analogues in hamster hepatocytes. These results indicate significant differences in the way in which cholesterol metabolism responds to cyclic AMP in cultured rat and hamster hepatocytes.     

Spectral analysis in cyclic changes of human sleep evaluation

In this study cyclic changes of human sleep structure were examined. For whole-night polysomnograms of 35 healthy volunteers of both sexes, manual hypnograms were created and divided into NREM-REM cycles. EEG signals from C3-A2 derivation were analysed by computer using a Fast Fourier Transform (FFT). For consecutive NREM-REM cycles of individual sleep stages, EEG power density contents for delta, theta, alpha, sigma and beta waves were analysed. For consecutive sleep cycles, a clear decrease in NREM sleep duration, especially slow wave sleep duration, was obtained. In addition, a decrease in power density of delta waves was observed. For consecutive sleep cycles, increases in REM sleep duration and in power density of theta and alpha waves were obtained. In consecutive sleep cycles, high amplitude delta slow waves are replaced by higher frequency and lower amplitude waves. Thus stages of NREM sleep are replaced by stages of REM.